Recently we developed a new type of RNA nanostructure that forms a truncated tetrahedron. The structure was built from our hexameric ring where 4 sides of the tetrahedral structure each contain the hexmeric ring, but each ring contains 3 H-shaped crossover connectors to the other rings. This type of construct allows for the incorporation of up to 12 functional entities such as Dicer substrates, beacons and/or aptamers. We found that cells seem to take up these constructs better than some of the other RNA nanoconstructs. The hypothesis that nanoparticle shape and size matter regarding functionality seems to be true. Due, at least in part, to the better uptake we found that knockdown of targeted genes to induce cell death, using incorporated Dicer substrate PLK1 is more efficacious than some of our other particles. Several different methods were used to verify the assembly of this particle including the newly acquired atomic force microscope (AFM). ---Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduced gaps into the double-stranded regions of the RNA rings. Molecular dynamics simulations were used to assess the dynamic behavior and the changes in the flexibility of the designs. The changes suggested by simulations, however, cannot be clearly confirmed by conventional techniques such as nondenaturing polyacrylamide gel electrophoresis and dynamic light scattering. Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of the new assemblies. To address these deficiencies, we introduced a computer-assisted quantification strategy, which is based on an algorithmic AFM-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validated this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis provided a broad-spectrum approach for the future development of nucleic acid-based nanotechnology. ---Several different computational and experimental protocols for the development and testing of RNA-based nanoparticles were published. The protocols included: 1) how a context sensitive RNA-based switch was designed and tested. Other switches sensitive to different cellular environments and targeting different genes could be designed using a similar protocol; 2) How oxime ether lipids were designed and used for the delivery of siRNAs was described including variations on the head groups and hydrophobic chains. Optimal configurations of these lipids were described that transfect their cargo in an efficient manner; 3) RNA nanoparticles can be made by single pot assembly protocols or by co-transcriptional assembly. The protocols for co-transcriptional assembly were described; 4) To gain important insights into the 3D molecular structure of RNA-based nanoparticles, molecular dynamics simulations are performed. The means for applying such a methodology were described; 5) A variety of designed RNA ring structures (ranging from triangles to hexagonal rings) have been reported in the scientific literature. Designing self-assembling RNA ring structures from structural motifs is, however, a nontrivial problem as there are many combinations of motifs and linking helices. Moreover, most combinations of motifs and linker helices will not lead to ring closure. A protocol for a web-browser based workflow for creating RNA rings using Galaxy, a web-based platform that can be used for workflow management was described. ---To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allow higher stability in blood serum, attachment of fluorescent markers for tracking, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. Diceable substrate siRNA could be split into two components, each consisting of an RNA/DNA hybrid. Complementary RNA single-stranded toeholds rather than DNA can be used in the construction of the hybrids. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and minimizing the design constraints of the resulting hybrid duplexes. RNA-based hybrids containing 3 Dicer substrate siRNAs for synergistic simultaneous targeting of apoptosis-related genes in HT29 tumors are now being used, after significant testing in cell cultures, in a comprehensive mouse study being funded, in part, by the Invention Development Program. Initial results look encouraging. --- Another study involving anaplastic thyroid cancer, in conjunction with the clinical center is progressing. In this case our RNA cubes containing 6 Dicer substrate arms are being used to synergistically target simultaneously 3 genes to induce apoptosis in the tumor cells. Cell culture experiments indicate that these particles are very potent in this cell line. ---Since we can control immune response with RNA-based nanoparticles, we have been collaborating with Joost Oppenheim- CCR, and Chris Jewell-UMD to take advantage of these properties to activate the immune system for anti-cancer treatment (funded in part by an UMD-NCI Partnership for Integrative Cancer Research grant). ---The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their negative charge. Thus, we are developing lipid and polymer formulations. In the case of the lipids we have constructed delivery agents consisting of DOTAP, DOPE and DSPE-PEG2000 to target cancer cells (in collaboration with Esta Sterneck, CCR). Experiments look quite positive. In addition, we are working with Jonathan Lovell (U of Buffalo) on the development of photoactivatable polymers for the delivery of our RNA-based nanoparticles. Results are very encouraging showing minimal leakage without laser treatment and significant functionality when laser treated. A second-generation chlorin-based photosensitizer, HPPH shows tremendous therapeutic potential in clinical trials in treatment of esophageal cancer. We, in collaboration with Sunil Dubey (Birla Institute of Technology & Science) have developed and validated a bioanalytical method for estimation of HPPH (a compound used in photodynamic therapy) in rat plasma using High Performance Liquid Chromatography with PDA detector. --Colon-26 mice using an HPPH LNP showed superior efficacy using PDT.--- A CRADA is in the final stages of negotiation with a startup company that was established out of the NCI Nanochallenge. The plan is for the company to use our RNA-based nanoparticles for glioblastoma. ---An invited book on protocols for RNA Nanobiology has recently been published, and in addition, invited review papers and book chapters were also written on the above described subjects.